This experimental work explores the application of an innovative, eco-friendly, and highly-reproducible nanofabrication technique, namely Pulse-Atomic Force Lithography (P-AFL), as the starting point of a process that employs eco-sustainable materials to manufacture nanowires. A thin film made of chitosan, a biopolymer, was obtained by spin-coating a solution of medium-molecular-weight chitosan at a concentration of 0.8% w/v in acetic acid 1% v/v on a silicon oxide substrate. The surface morphology and thickness of the resulting chitosan film was characterized by Atomic Force Microscopy (AFM): the film was homogeneous, with a roughness of about 1 nm and a thickness of about 52 nm. Then, the P-AFL technique was optimized to pattern a set of nanogrooves on chitosan. A metal layer of chrome and titanium, with a 1:10 ratio, was deposited on the chitosan film by Electron Bem Evaporation (EBE). Successively, the samples were submitted to a lift-off process by an acetic acid solution (1% v/v) with the aim of dissolving the chitosan layer. The resulting nanowires were then characterized by AFM and Scanning Electron Microscopy (SEM): the nanostructures appeared well-fabricated, with a length of 10 μm and a height of (71 ± 20) nm. The entire process is based on the use of the P-AFL technique, which does not require the use of toxic chemicals, i.e., the developer resists for conventional optical lithography. Moreover, chitosan is eco-sustainable, as it is derived from natural sources, bio-compatible, and bio-degradable, and the lift-off step is performed by using acetic acid, a non-harmful chemical.